Single-particle tracking (SPT) has enabled the direct observation of dynamic behaviors of particles (as used herein, a particle can be a single biomolecule, a molecular complex, a vesicle, a lipid granule, or a viral capsid) inside complex biological systems [1], with particle localization precision better than the diffraction limit of light [2,3]. Through trajectory analysis SPT has provided insight into motor protein kinetics [4,5] cellular membrane dynamics, [6-8] mRNA transport [9,10] and virus internalization processes [11,12]. As the basis of passive micro rheology, SPT has also shed light on the local environments of tracked particles through the observation of changes in particles' random movements [13].
Whereas SPT is becoming a powerful research tool, all current techniques suffer from one or more of the following problems: shallow penetration depth (arising from the use of one-photon excitation) [14-16], limited ztracking range (e.g. TIRF microscopy), poor temporal resolution (e.g. frame-by-frame analysis in camera-based methods) [17,18], and low information content (e.g. no information on the fluorescence lifetime) [19]. As two-photon (2P) microscopy has become a standard method for deep tissue imaging [20] a few reports demonstrated 3D tracking based on two-photon excitation. The first approach used a circular scanning pattern of the focused laser beam to track particles [2, 21] but was limited to a temporal resolution of 20˜32 ms due to the laser scanning and signal demodulation. More recently, 3D tracking of gold nanorods with 2P excitation was demonstrated by exciting multiple foci and detecting fluorescence with an EM-CCD [18] but the 3D temporal resolution was limited to about 0.5 s. Moreover, the use of a camera in multifocal 2P laser scanning microscopy (2P-LSM) limits the working depth of SPT in scattering samples [22]. Although SPT with superior temporal resolution (bounded mainly by the emission rate of the fluorescent label) and simultaneous fluorescence lifetime measurements have been achieved using confocal setups with 3-5 single-element/photon-counting detectors (PMTs or APDs) for spatial filtering [14-16], these methods not only have limited working depth (not using 2P excitation for tracking) but also suffer from loss of signals due to the non-overlapping excitation and collection efficiency peaks in spatial filtering. Currently there is no single solution to all of the above issues.
Molecular trafficking within cells, tissues, and engineered 3D multicellular models is critical to the understanding of the development and treatment of various diseases including cancer. However, current tracking methods are either confined to two dimensions or limited to an interrogation depth of about 15 μm.
A 3D tracking method is presented herein capable of quantifying rapid molecular transport dynamics in highly scattering environments at depths up to 200 μm. The temporal resolution and a spatial localization precision as good as 50 μs and 35 nm have been verified. Built upon spatiotemporally multiplexed two-photon excitation, this approach requires only one detector for 3D particle tracking and allows for two-photon, multicolor imaging. 3D tracking of EGFR complexes at a depth of approximately 100 μm in tumor spheroids is presented herein.
Embodiments disclosed herein, coined TSUNAMI (Tracking Single-particles Using Nonlinear And Multiplexed Illumination), are capable of tracking particles at depths up to 200 μm in scattering samples with 22/90 [xy/z] nm spatial localization precision and 50 μs temporal resolution. At shallow depths the localization precision can be as good as 35 nm in all three dimensions. The approach is based on passive pulse splitters used for nonlinear microscopy to achieve spatiotemporally multiplexed 2P excitation and temporally demultiplexed detection to discern the 3D position of the particle. The z-tracking range is up to approximately 50 μm (limited by the objective z-piezo stage) and the method enables simultaneous fluorescence lifetime measurements on the tracked particles. A major advantage of this method over previous tracking approaches is that it requires only one detector for SPT and is compatible with multi-color two-photon microscopy.
The technology uses a unique spatiotemporally multiplexed point-spread function (PSF). Traditional PSFs are circular and comprised of a single excitation beam. This novel PSF uses 4 temporally offset beams (offset in 3.3 ns increments in particular embodiments) that illuminate a tetrahedral pattern in image space. This PSF is achieved by a passive optical system placed before the scanning optics of a traditional two-photon microscope. In certain embodiments, the tracking algorithm utilizes Time-Correlated-Single-Photon Counting (TCSPC) electronics (Becker & Hickl SPC-150) with 19.5 ps time resolution to demultiplex the fluorescent decay from 4 excitation beams and yield the signal that can be processed to achieve sub-diffraction localization of a single particle. To track the particle with the localization signal galvanometric mirrors (xy scanning) and an objective focusing piezo stage (z-dimension) actuate.